Structural Biology of the Apical Bile Acid Transporter

描述(申请人提供)：心尖部钠依赖胆汁酸转运体(ASBT)在胆盐的肠-肝循环、胆固醇稳态中起关键作用，也是高胆固醇药物的分子靶点。虽然转运蛋白的序列已知，但对其膜的拓扑结构仍存在争议，对ASBT的结构与功能以及配体结合域的了解也很少。拟议的研究将集中在ASBT的结构生物学上。利用一种结合了分子和计算生物学的新方法，我们的长期目标是描绘ASBT的三维结构、配体结合结构域和细胞转运机制。具体目标如下：(1)使用一系列拓扑扫描方法确定ASBT的膜拓扑；(2)构建ASBT的全面结构和预测模型，可以将结构点突变与配体亲和力和转运的变化联系起来；(3)通过定点突变定义ASBT的功能区；我们开发了一种计算机辅助的定点突变方法，以探测ASBT蛋白中与配体和钠相互作用有关的氨基酸残基；(4)通过质谱学确定ASBT的配体和结合域；我们将使用选择性光亲和标记来确定配体结合肽序列。通过这些研究获得的信息将大大增加我们对驱动胆汁酸运输的结构相互作用的理解，并进一步加深我们对溶质载体蛋白的一般结构知识。此外，它可能有助于未来针对高胆固醇血症和相关心血管疾病的特定治疗策略的开发。

DESCRIPTION (provided by applicant): The apical sodium-dependent bile acid transporter (ASBT) plays a key role in the enterohepatic recycling of bile salts, cholesterol homeostasis, and serves as a molecular target for hypercholesterolemic agents. Although the transporter sequence is known, there is controversy about its membrane topology and very little is known about ASBT structure-function and ligand binding domains. The proposed research will focus on the structural biology of ASBT. Using a novel approach that combines molecular and computational biology our long-term goal is to delineate the three-dimensional structure, ligand-binding domains, and cellular transport mechanism of ASBT. The following specific aims will be addressed: (1) define the membrane topology of ASBT using a series of topology scanning approaches; (2) Construct a comprehensive structural and predictive model of ASBT that can correlate structural point mutations to changes in ligand affinity and transport; (3) Define the functional regions of ASBT by site-directed mutagenesis; we have developed a computer-assisted site-directed mutagenesis approach to probe ASBT protein for amino acid residues implicated in ligand and sodium interactions; (4) Determine the ligand binding domains of ASBT by mass spectrometry; we will employ selective photoaffinity labels to determine ligand-binding peptide sequences. Information gained by these studies will significantly increase our understanding of the structural interactions that drive bile acid transport and further our structural knowledge of solute carrier proteins in general. Additionally, it may aid future development of specific therapeutic strategies against hypercholesterolemia and related cardiovascular diseases.